Vaccines against multiple subtypes of influenza virus

ABSTRACT

An aspect of the present invention is directed towards DNA plasmid vaccines capable of generating in a mammal an immune response against a plurality of influenza virus subtypes, comprising a DNA plasmid and a pharmaceutically acceptable excipient. The DNA plasmid is capable of expressing a consensus influenza antigen in a cell of the mammal in a quantity effective to elicit an immune response in the mammal, wherein the consensus influenza antigen comprises consensus hemagglutinin (HA), neuraminidase (NA), matrix protein, nucleoprotein, M2 ectodomain-nucleo-protein (M2e-NP), or a combination thereof. Preferably the consensus influenza antigen comprises HA, NA, M2e-NP, or a combination thereof. The DNA plasmid comprises a promoter operably linked to a coding sequence that encodes the consensus influenza antigen. Additionally, an aspect of the present invention includes methods of eliciting an immune response against a plurality of influenza virus subtypes in a mammal using the DNA plasmid vaccines provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/987,284, filed Nov. 12, 2007, the contents of which areincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to improved influenza vaccines, improvedmethods for inducing immune responses, and for prophylactically and/ortherapeutically immunizing individuals against influenza.

BACKGROUND

The use of nucleic acid sequences to vaccinate against animal and humandiseases has been studied. Studies have focused on effective andefficient means of delivery in order to yield necessary expression ofthe desired antigens, resulting immunogenic response and ultimately thesuccess of this technique. One method for delivering nucleic acidsequences such as plasmid DNA is the electroporation (EP) technique. Thetechnique has been used in human clinical trials to deliver anti-cancerdrugs, such as bleomycin, and in many preclinical studies on a largenumber of animal species.

The influenza virus genome is contained on eight single (non-paired) RNAstrands that code for eleven proteins (HA, NA, NP, M1, M2, NS1, NEP, PA,PB1, PB1-F2, PB2). The segmented nature of the genome allows for theexchange of entire genes between different viral strains during cellularcohabitation. The eight RNA segments are: HA, which encodeshemagglutinin (about 500 molecules of hemagglutinin are needed to makeone virion); NA, which encodes neuraminidase (about 100 molecules ofneuraminidase are needed to make one virion); NP, which encodesnucleoprotein; M, which encodes two matrix proteins (the M1 and the M2)by using different reading frames from the same RNA segment (about 3000matrix protein molecules are needed to make one virion); NS, whichencodes two distinct non-structural proteins (NS1 and NEP) by usingdifferent reading frames from the same RNA segment; PA, which encodes anRNA polymerase; PB1, which encodes an RNA polymerase and PB1-F2 protein(induces apoptosis) by using different reading frames from the same RNAsegment; and PB2, which encodes an RNA polymerase.

Influenza hemagglutinin (HA) is expressed on the surface of influenzaviral particles and is responsible for initial contact between the virusand its host cell. HA is a well-known immunogen. Influenza A strainH5N1, an avian influenza strain, particularly threatens the humanpopulation because of its HA protein (H5) which, if slightly geneticallyreasserted by natural mutation, has greatly increased infectivity ofhuman cells as compared to other strains of the virus. Infection ofinfants and older or immunocompromised adult humans with the viral H5N1strain is often correlated to poor clinical outcome. Therefore,protection against the H5N1 strain of influenza is a great need for thepublic.

There are two classes of anti-influenza agents available, inhibitors ofinfluenza A cell entry/uncoating (such as antivirals amantadine andrimantadine) and neuraminidase inhibitors (such as antiviralsoseltamivir, zanamivir). These antiviral agents inhibit the cellularrelease of both influenza A and B. Concerns over the use of these agentshave been reported due to findings of strains of virus resistant tothese agents.

Influenza vaccines are a popular seasonal vaccine and many people haveexperienced such vaccinations. However, the vaccinations are limited intheir protective results because the vaccines are specific for certainsubtypes of virus. The Centers for Disease Control and Preventionpromote vaccination with a “flu shot” that is a vaccine that containsthree influenza viruses (killed viruses): one A (H3N2) virus, one A(H1N1) virus, and one B virus. They also report that the viruses in thevaccine change each year based on international surveillance andscientists' estimations about which types and strains of viruses willcirculate in a given year. Thus, it is apparent that vaccinations arelimited to predictions of subtypes, and the availability of a specificvaccine to that subtype.

There still remains a need for effective influenza vaccines that areeconomical and effective across numerous subtypes. Further, thereremains a need for an effective method of administering DNA vaccines toa mammal in order to provide immunization against influenza eitherprophylatically or therapeutically.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a DNA plasmid vaccinecapable of generating in a mammal an immune response against a pluralityof influenza virus subtypes, comprising a DNA plasmid and apharmaceutically acceptable excipient. The DNA plasmid is capable ofexpressing a consensus influenza antigen in a cell of the mammal in aquantity effective to elicit an immune response in the mammal, whereinthe consensus influenza antigen comprises consensus hemagglutinin (HA),neuraminidase (NA), matrix protein, nucleoprotein, M2ectodomain-nucleo-protein (M2e-NP), or a combination thereof. Preferablythe consensus influenza antigen comprises HA, NA, M2e-NP, or acombination thereof. The DNA plasmid comprises a promoter operablylinked to a coding sequence that encodes the consensus influenzaantigen. Preferably, the DNA plasmid vaccine is one having aconcentration of total DNA plasmid of 1 mg/ml or greater.

Another aspect of the present invention relates to DNA plasmids capableof expressing a consensus influenza antigen in a cell of the mammal, theconsensus influenza antigen comprising consensus hemagglutinin (HA),neuraminidase (NA), matrix protein, nucleoprotein, M2ectodomain-nucleo-protein (M2e-NP), or a combination thereof. Preferablythe consensus influenza antigen comprises HA, NA, M2e-NP, or acombination thereof. The DNA plasmid comprises a promoter operablylinked to a coding sequence that encodes the consensus influenzaantigen.

Another aspect of the present invention relates to methods of elicitingan immune response against a plurality of influenza virus subtypes in amammal. The methods include delivering a DNA plasmid vaccine to tissueof the mammal, the DNA plasmid vaccine comprising a DNA plasmid capableof expressing a consensus influenza antigen in a cell of the mammal toelicit an immune response in the mammal, the consensus influenza antigencomprising consensus HA, NA, M2e-NP or a combination thereof, andelectroporating cells of the tissue with a pulse of energy at a constantcurrent effective to permit entry of the DNA plasmids in the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The numerous objects and advantages of the present invention may bebetter understood by those skilled in the art by reference to theaccompanying figures, in which:

FIG. 1 displays a schematic representation (plasmid maps) of the DNAplasmid constructs used in the studies described herein. Consensus HA,NA and M2e-NP constructs were generated by analyzing primary virussequences from 16 H5 viruses that have proven fatal to humans in recentyears, and over 40 human N1 viruses (Los Alamos National Laboratory'sInfluenza Sequence Database). After generating the consensus sequences,the constructs were optimized for mammalian expression, including theaddition of a Kozak sequence, codon optimization, and RNA optimization.These constructs were then subcloned into the pVAX vector (Invitrogen,Carlsbad, Calif.). Plasmids pGX2001 (consensus HA), pGX2002 (consensusNA), pGX2003 (consensus M2e-NP) are shown. The plasmid pCMVSEAP,displayed, encodes the reporter protein secreted embryonic alkalinephosphatase (SEAP).

FIG. 2 displays a bar graph of the results of the HI titers in pig serumat Day 35 post-injection. The highest titers were found in the groupadministered 2 mg of HA-expressing plasmid at a current setting of 0.5 A(120±40; *P=0.11 versus 2 mg/0.3 A and *P=0.02 versus 2 mg/0.1 A). Thethree groups administered descending doses of plasmid and electroporatedat 0.5 A also demonstrated decreasing HI titers.

FIG. 3 displays a bar graph of the IFN-γ ELISpot counts. The counts werehighest in pigs administered 2 mg of HA and 2 mg of NA plasmid vaccine(for a total of 4 mg plasmid) and electroporated with 0.3 A of current(2000 spots) and in the group administered 0.8 mg of HA and 0.8 mg of NAplasmid vaccine (for a total of 1.6 mg plasmid) electroporated with 0.5A of current (934 spots). For comparison purposes, the cellular immuneresponses of an unimmunized control group are depicted.

FIGS. 4A and 4B display bar graphs showing results from muscle biopsiesfrom treated pigs at Day 14 and Day 35: FIG. 4A displays a bar graphshowing the mean pathology scores for all groups. FIG. 4B displays a bargraph showing the muscle necrosis and fibrosis scores. The groupinjected with 6 mg total plasmid and electroporated at 0.5 A exhibitedthe highest mean pathology score (*P<0.0002 as compared to controls).The pathology scores were significantly reduced by Day 35 compared toDay 14 in all groups (P<0.05) except for the 0.3 mg/0.3 A group(P=0.057) and 2.4 mg/0.1 A group (P=1.0).

FIG. 5 displays the percent change in weight of ferrets after challengewith H5N1 virus (A/Viet/1203/2004(H5N1)/PR8-IBCDC-RG). Ferrets that werevaccinated with HA, HA+M2e-NP or HA+M2e-NP+NA lost significantly lessweight than control animals (*P<0.005 versus controls) in the 9 dayspost-challenge period. One animal in the HA vaccine group actuallygained weight post-challenge.

FIG. 6 displays a graph showing the body temperatures of ferrets duringthe 9 days post-challenge. Control animals showed higher bodytemperatures than the vaccinated animals. The body temperature on day 5is not depicted as it was measured at a different time of day and allthe temperatures regardless of group were lower.

FIG. 7 displays a bar graph of results from HI titers in ferrets aftervaccination; the assay was performed using reassortant viruses obtainedfrom the Center for Disease Control: A/Viet/1203/04 or Indo/05/2005influenza strains.

FIG. 8 displays a bar graph of results from HI titers measured threeweeks after the second immunization. Macaques immunized ID followed byEP showed significantly higher HI titers than all other groups (P<0.03).Non-treated controls did not exhibit any HI titers.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following abbreviated, or shortened, definitions are given to helpthe understanding of the preferred embodiments of the present invention.The abbreviated definitions given here are by no means exhaustive norare they contradictory to the definitions as understood in the field ordictionary meaning. The abbreviated definitions are given here tosupplement or more clearly define the definitions known in the art.

DEFINITIONS

Sequence homology for nucleotides and amino acids as used herein may bedetermined using FASTA, BLAST and Gapped BLAST (Altschul et al., Nuc.Acids Res., 1997, 25, 3389, which is incorporated herein by reference inits entirety) and PAUP* 4.0b10 software (D.L. Swofford, SinauerAssociates, Massachusetts). Briefly, the BLAST algorithm, which standsfor Basic Local Alignment Search Tool is suitable for determiningsequence similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403-410,which is incorporated herein by reference in its entirety). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. One measure of similarity providedby the BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide sequences would occur by chance. For example, a nucleic acidis considered similar to another if the smallest sum probability incomparison of the test nucleic acid to the other nucleic acid is lessthan about 1, preferably less than about 0.1, more preferably less thanabout 0.01, and most preferably less than about 0.001. “Percentage ofsimilarity” can be calculated using PAUP* 4.0b10 software (D.L.Swofford, Sinauer Associates, Massachusetts). The average similarity ofthe consensus sequence is calculated compared to all sequences in thephylogenic tree.

As used herein, the term “genetic construct” or “nucleic acid construct”is used interchangeably and refers to the DNA or RNA molecules thatcomprise a nucleotide sequence which encodes protein. The codingsequence, or “encoding nucleic acid sequence,” includes initiation andtermination signals operably linked to regulatory elements including apromoter and polyadenylation signal capable of directing expression inthe cells of the individual to whom the nucleic acid molecule isadministered.

As used herein, the term “expressible form” refers to nucleic acidconstructs that contain the necessary regulatory elements operablelinked to a coding sequence that encodes a protein such that whenpresent in the cell of the individual, the coding sequence will beexpressed.

The term “constant current” is used herein to define a current that isreceived or experienced by a tissue, or cells defining said tissue, overthe duration of an electrical pulse delivered to same tissue. Theelectrical pulse is delivered from the electroporation devices describedherein. This current remains at a constant amperage in said tissue overthe life of an electrical pulse because the electroporation deviceprovided herein has a feedback element, preferably having instantaneousfeedback. The feedback element can measure the resistance of the tissue(or cells) throughout the duration of the pulse and cause theelectroporation device to alter its electrical energy output (e.g.,increase voltage) so current in same tissue remains constant throughoutthe electrical pulse (on the order of microseconds), and from pulse topulse. In some embodiments, the feedback element comprises a controller.

The term “feedback” or “current feedback” is used interchangeably andmeans the active response of the provided electroporation devices, whichcomprises measuring the current in tissue between electrodes andaltering the energy output delivered by the EP device accordingly inorder to maintain the current at a constant level. This constant levelis preset by a user prior to initiation of a pulse sequence orelectrical treatment. Preferably, the feedback is accomplished by theelectroporation component, e.g., controller, of the electroporationdevice, as the electrical circuit therein is able to continuouslymonitor the current in tissue between electrodes and compare thatmonitored current (or current within tissue) to a preset current andcontinuously make energy-output adjustments to maintain the monitoredcurrent at preset levels. In some embodiments, the feedback loop isinstantaneous as it is an analog closed-loop feedback.

The terms “electroporation,” “electro-permeabilization,” or“electro-kinetic enhancement” (“EP”) as used interchangeably hereinrefer to the use of a transmembrane electric field pulse to inducemicroscopic pathways (pores) in a bio-membrane; their presence allowsbiomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, andwater to pass from one side of the cellular membrane to the other.

The term “decentralized current” is used herein to define the pattern ofelectrical currents delivered from the various needle electrode arraysof the electroporation devices described herein, wherein the patternsminimize, or preferably eliminate, the occurrence of electroporationrelated heat stress on any area of tissue being electroporated.

The term “feedback mechanism” as used herein refers to a processperformed by either software or hardware (or firmware), which processreceives and compares the impedance of the desired tissue (before,during, and/or after the delivery of pulse of energy) with a presentvalue, preferably current, and adjusts the pulse of energy delivered toachieve the preset value. The term “impedance” is used herein whendiscussing the feedback mechanism and can be converted to a currentvalue according to Ohm's law, thus enabling comparisons with the presetcurrent. In a preferred embodiment, the “feedback mechanism” isperformed by an analog closed loop circuit.

The term “immune response” is used herein to mean the activation of ahost's immune system, e.g., that of a mammal, in response to theintroduction of influenza consensus antigen via the provided DNA plasmidvaccines. The immune response can be in the form of a cellular orhumoral response, or both.

The term “consensus” or “consensus sequence” is used herein to mean asynthetic nucleic acid sequence, or corresponding polypeptide sequence,constructed based on analysis of an alignment of multiple subtypes of aparticular influenza antigen, that can be used to induce broad immunityagainst multiple subtypes or serotypes of a particular influenzaantigen. Consensus influenza antigens include HA, including consensusH1, H2, H3, or H5, NA, NP, matrix protein, and nonstructural protein.Also, synthetic antigens such as fusion proteins, e.g., M2e-NP, can bemanipulated to consensus sequences (or consensus antigens).

The term “adjuvant” is used herein to mean any molecule added to the DNAplasmid vaccines described herein to enhance antigenicity of theinfluenza antigen encoded by the DNA plasmids and encoding nucleic acidsequences described hereinafter.

The term “subtype” or “serotype” is used herein interchangeably and inreference to influenza viruses, and means genetic variants of aninfluenza virus antigen such that one subtype is recognized by an immunesystem apart from a different subtype (or, in other words, each subtypeis different in antigenic character from a different subtype).

In some embodiments, there are DNA plasmids capable of expressing aconsensus influenza antigen in a cell of the mammal, the consensusinfluenza antigen comprising consensus hemagglutinin (HA), neuraminidase(NA), matrix protein, nucleoprotein, M2 ectodomain-nucleo-protein(M2e-NP), or a combination thereof. Preferably the consensus influenzaantigen comprises HA, NA, M2e-NP, or a combination thereof. The DNAplasmid comprises a promoter operably linked to a coding sequence thatencodes the consensus influenza antigen.

In some embodiments, the present invention provides DNA plasmid vaccinesthat are capable of generating in a mammal an immune response against aplurality of influenza virus subtypes, the DNA plasmid vaccinescomprising a DNA plasmid and a pharmaceutically acceptable excipient.The DNA plasmid is capable of expressing a consensus influenza antigenin a cell of the mammal in a quantity effective to elicit an immuneresponse in the mammal, wherein the consensus influenza antigencomprises consensus hemagglutinin (HA), neuraminidase (NA), matrixprotein, nucleoprotein, M2 ectodomain-nucleo-protein (M2e-NP), or acombination thereof. Preferably the consensus influenza antigencomprises HA, NA, M2e-NP, or a combination thereof. The DNA plasmidcomprises a promoter operably linked to a coding sequence that encodesthe consensus influenza antigen. In some embodiments, the DNA plasmidvaccine is one having a concentration of total DNA plasmid of 1 mg/ml orgreater. The immune response can be a cellular or humoral response, orboth; preferably, the immune response is both cellular and humoral.

In some embodiments, the DNA plasmid can further include an IgG leadersequence attached to an N-terminal end of the coding sequence andoperably linked to the promoter. In addition, in some embodiments, theDNA plasmid can further include a polyadenylation sequence attached tothe C-terminal end of the coding sequence. In some embodiments, the DNAplasmid is codon optimized.

In some embodiments of the present invention, the DNA plasmid vaccinescan further include an adjuvant. In some embodiments, the adjuvant isselected from the group consisting of: alpha-interferon,gamma-interferon, platelet derived growth factor (PDGF), TNFα, TNFβ,GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attractingchemokine (CTACK), epithelial thymus-expressed chemokine (TECK),mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80,CD86 including IL-15 having the signal sequence deleted and optionallyincluding the signal peptide from IgE. Other genes which may be usefuladjuvants include those encoding: MCP-1, MIP-1α, MIP-1p, IL-8, RANTES,L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1,VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor,fibroblast growth factor, IL-7, nerve growth factor, vascularendothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1,DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2,DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88,IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon responsegenes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4,RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B,NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof. Insome preferred embodiments, the adjuvant is selected from IL-12, IL-15,CTACK, TECK, or MEC.

In some embodiments, the pharmaceutically acceptable excipient is atransfection facilitating agent, which can include the following:surface active agents, such as immune-stimulating complexes (ISCOMS),Freunds incomplete adjuvant, LPS analog including monophosphoryl lipidA, muramyl peptides, quinone analogs, vesicles such as squalene andsqualene, hyaluronic acid, lipids, liposomes, calcium ions, viralproteins, polyanions, polycations, or nanoparticles, or other knowntransfection facilitating agents. Preferably, the transfectionfacilitating agent is a polyanion, polycation, includingpoly-L-glutamate (LGS), or lipid. Preferably, the transfectionfacilitating agent is poly-L-glutamate, and more preferably, thepoly-L-glutamate is present in the DNA plasmid vaccine at aconcentration less than 6 mg/ml. In some embodiments, the concentrationof poly-L-glutamate in the DNA plasmid vaccine is less than 4 mg/ml,less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than0.500 mg/ml, less than 0.250 mg/ml, less than 0.100 mg/ml, less than0.050 mg/ml, or less than 0.010 mg/ml.

In some embodiments, the DNA plasmid vaccine can include a plurality ofdifferent DNA plasmids. In some examples, the different DNA plasmidsinclude a DNA plasmid comprising a nucleic acid sequence that encodes aconsensus HA, a DNA plasmid comprising a sequence that encodes aconsensus NA, and a DNA plasmid comprising a sequence that encodes aconsensus M2e-NP. In some embodiments, the consensus HA is a consensusH1, consensus H2, consensus H3, or consensus H5. Preferably, theconsensus HA is nucleotide sequence that is SEQ ID NO:1 (H5N1 HAconsensus DNA), SEQ ID NO:9 (consensus H1 DNA), SEQ ID NO: 11 (consensusH3 DNA), or SEQ ID NO:13 (consensus H5). The consensus HA can also be anucleotide sequence encoding a polypeptide of the sequence SEQ ID NO: 2,SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. In some embodiments, theconsensus NA is a nucleotide sequence that is SEQ ID NO: 3, or anucleotide sequence encoding a polypeptide of the sequence SEQ ID NO: 4.In some embodiments, the consensus M2e-NP is a nucleotide sequence thatis SEQ ID NO:7, or a nucleotide sequence encoding a polypeptide of thesequence SEQ ID NO:8. In one preferred embodiment, the DNA plasmidvaccine includes a DNA plasmid comprising a sequence that encodes aconsensus H1, a DNA plasmid comprising a sequence that encodes aconsensus H2, a DNA plasmid comprising a sequence that encodes aconsensus H3, a DNA plasmid comprising a sequence that encodes aconsensus H5, a DNA plasmid comprising a sequence that encodes aconsensus NA, and a DNA plasmid comprising a sequence that encodes aconsensus M2e-NP.

In some embodiments, the DNA plasmid vaccine can include a plurality ofdifferent DNA plasmids, including at least one DNA plasmid that canexpress consensus influenza antigens and at least one that can expressone influenza subtype antigen. In some examples, the different DNAplasmids that express consensus antigen include a DNA plasmid comprisinga nucleic acid sequence that encodes a consensus HA, a DNA plasmidcomprising a sequence that encodes a consensus NA, and a DNA plasmidcomprising a sequence that encodes a consensus M2e-NP. In someembodiments, the DNA plasmid vaccine comprises a DNA plasmid that canexpress a consensus HA antigen, e.g., consensus H1, H3 or H5, and a DNAplasmid that can express any one of the following influenza A antigens:H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16,N1, N2, N3, N4, N5, N6, N7, N8, N9, NP, M1, M2, NS1, or NEP, or acombination thereof. In some embodiments, the DNA plasmid vaccinecomprises a DNA plasmid that can express a consensus NA antigen and aDNA plasmid that can express any one of the following influenza Aantigens: H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, H16, N1, N2, N3, N4, N5, N6, N7, N8, N9, NP, M1, M2, NS1, or NEP,or a combination thereof. In some embodiments, the DNA plasmid vaccinecomprises a DNA plasmid that can express a consensus M2e-NP and a DNAplasmid that can express any one of the following influenza A antigens:H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16,N1, N2, N3, N4, N5, N6, N7, N8, N9, NP, M1, M2, NS1, or NEP, or acombination thereof.

In some embodiments, the DNA plasmid vaccine can be delivered to amammal to elicit an immune response; preferably the mammal is a primate,including human and nonhuman primate, a cow, pig, chicken, dog, orferret. More preferably, the mammal is a human primate.

One aspect of the present invention relates to methods of eliciting animmune response against a plurality of influenza virus subtypes in amammal. The methods include delivering a DNA plasmid vaccine to tissueof the mammal, the DNA plasmid vaccine comprising a DNA plasmid capableof expressing a consensus influenza antigen in a cell of the mammal toelicit an immune response in the mammal, the consensus influenza antigencomprising consensus HA, NA, M2e-NP or a combination thereof, andelectroporating cells of the tissue with a pulse of energy at a constantcurrent effective to permit entry of the DNA plasmids in the cells.

In some embodiments, the methods of the present invention include thedelivering step, which comprises injecting the DNA plasmid vaccine intointradermic, subcutaneous or muscle tissue. Preferably, these methodsinclude using an in vivo electroporation device to preset a current thatis desired to be delivered to the tissue; and electroporating cells ofthe tissue with a pulse of energy at a constant current that equals thepreset current. In some embodiments, the electroporating step furthercomprises: measuring the impedance in the electroporated cells;adjusting energy level of the pulse of energy relative to the measuredimpedance to maintain a constant current in the electroporated cells;wherein the measuring and adjusting steps occur within a lifetime of thepulse of energy.

In some embodiments, the electroporating step comprises delivering thepulse of energy to a plurality of electrodes according to a pulsesequence pattern that delivers the pulse of energy in a decentralizedpattern.

In some embodiments, the DNA plasmid influenza vaccines of the inventioncomprise nucleotide sequences that encode a consensus HA, or a consensusHA and a nucleic acid sequence encoding influenza proteins selected fromthe group consisting of: SEQ ID NOS: 4, 6, and 8. SEQ ID NOS:1 and 13comprise the nucleic acid sequence that encodes consensus H5N1 HA and H5of influenza virus, respectively. SEQ ID NOS:2 and 14 comprise the aminoacid sequence for H5N1 HA and H5 of influenza virus, respectively. Insome embodiments of the invention, the vaccines of the inventioncomprise SEQ ID NO:3 or SEQ ID NO:4. SEQ ID NO:3 comprises the nucleicacid sequence that encodes influenza H1N1 and H5N1 (H1N1/H5N1) NAconsensus sequences. SEQ ID NO:4 comprises the amino acid sequence forinfluenza H1N1/H5N1 NA consensus sequences. In some embodiments of theinvention, the vaccines of the invention comprise SEQ ID NO:5 or SEQ IDNO:6. SEQ ID NO:5 comprises the nucleic acid sequence that encodesinfluenza H1N1/H5N1 M1 consensus sequences. SEQ ID NO:6 comprises theamino acid sequence for influenza H1N1/H5N1 M1 consensus sequences. Insome embodiments of the invention, the vaccines of the inventioncomprise SEQ ID NO:7 or SEQ ID NO:8. SEQ ID NO:7 comprises the nucleicacid sequence that encodes influenza H5N1 M2E-NP consensus sequence. SEQID NO:8 comprises the amino acid sequence for influenza H5N1 M2E-NPconsensus sequence. In some embodiments of the invention, the vaccinesof the invention comprise SEQ ID NO:9 or SEQ ID NO:10. SEQ ID NO:9comprises the nucleic acid sequence that encodes influenza H1N1 HAconsensus sequences. SEQ ID NO:4 comprises the amino acid sequence forinfluenza H1N1 HA consensus sequences. In some embodiments of theinvention, the vaccines of the invention comprise SEQ ID NO: 11 or SEQID NO: 12. SEQ ID NO:11 comprises the nucleic acid sequence that encodesinfluenza H3N1 HA consensus sequences. SEQ ID NO: 12 comprises the aminoacid sequence for influenza H3N1 HA consensus sequences. The consensussequence for influenza virus strain H5N1 HA includes the immunodominantepitope set forth in SEQ ID NO:1 or SEQ ID NO:13. The influenza virusH5N1 HA amino acid sequence encoded by SEQ ID NO: 1 is SEQ ID NO:2, andthat encoded by SEQ ID NO:13 is SEQ ID NO:14. The consensus sequence forinfluenza virus H1N1/H5N1 NA includes the immunodominant epitope setforth in SEQ ID NO:3. The influenza virus strains H1N1/H5N1 NA aminoacid sequence encoded by SEQ ID NO:3 is SEQ ID NO:4. The consensussequence for influenza virus strains H1N1/H5N1 M1 includes theimmunodominant epitope set forth in SEQ ID NO:5. The influenza virusH1N1/H5N1 M1 amino acid sequence encoded by SEQ ID NO:5 is SEQ ID NO:6.The consensus sequence for influenza virus H5N1 M2E-NP includes theimmunodominant epitope set forth in SEQ ID NO:7. The influenza virusH5N1 M2E-NP amino acid sequence encoded by SEQ ID NO:7 is SEQ ID NO:8.Vaccines of the present invention may include protein products encodedby the nucleic acid molecules defined above or any fragments ofproteins.

The present invention also comprises DNA fragments that encode apolypeptide capable of eliciting an immune response in a mammalsubstantially similar to that of the non-fragment for at least oneinfluenza subtype. The DNA fragments are fragments selected from atleast one of the various encoding nucleotide sequences of the presentinvention, including SEQ ID NOS:1, 3, 5, 7, 9, 11, and 13, and can beany of the following described DNA fragments, as it applies to thespecific encoding nucleic acid sequence provided herein. In someembodiments, DNA fragments can comprise 30 or more, 45 or more, 60 ormore, 75 or more, 90 or more, 120 or more, 150 or more, 180 or more, 210or more, 240 or more, 270 or more, 300 or more, 360 or more, 420 ormore, 480 or more, 540 or more, 600 or more, 660 or more, 720 or more,780 or more, 840 or more, 900 or more, 960 or more, 1020 or more, 1080or more, 1140 or more, 1200 or more, 1260 or more, 1320 or more, 1380 ormore, 1440 or more, 1500 or more, 1560 or more, 1620 or more, 1680 ormore, or 1740 or more nucleotides. In some embodiments, DNA fragmentscan comprise coding sequences for the immunoglobulin E (IgE) leadersequences. In some embodiments, DNA fragments can comprise fewer than60, fewer than 75, fewer than 90, fewer than 120, fewer than 150, fewerthan 180, fewer than 210, fewer than 240, fewer than 270, fewer than300, fewer than 360, fewer than 420, fewer than 480, fewer than 540,fewer than 600, fewer than 660, fewer than 720, fewer than 780, fewerthan 840, fewer than 900, fewer than 960, fewer than 1020, fewer than1080, fewer than 1140, fewer than 1200, fewer than 1260, fewer than1320, fewer than 1380, fewer than 1440, fewer than 1500, fewer than1560, fewer than 1620, fewer than 1680, or fewer than 1740 nucleotides.Preferably, the DNA fragments are fragments of SEQ ID NOS:1, 3, 7, 9, 11or 13, and more preferably fragments of SEQ ID NOS:1, 5, 9, 11, or 13,and even more preferably fragments of SEQ ID NOS:1, 9, or 13.

The present invention also comprises polypeptide fragments that arecapable of eliciting an immune response in a mammal substantiallysimilar to that of the non-fragment for at least one influenza subtype.The polypeptide fragments are selected from at least one of the variouspolypeptide sequences of the present invention, including SEQ ID NOS:2,4, 6, 8, 10, 12, and 14, and can be any of the following describedpolypeptide fragments, as it applies to the specific polypeptidesequence provided herein. In some embodiments, polypeptide fragments cancomprise 15 or more, 30 or more, 45 or more, 60 or more, 75 or more, 90or more, 105 or more, 120 or more, 150 or more, 180 or more, 210 ormore, 240 or more, 270 or more, 300 or more, 360 or more, 420 or more,480 or more, 540 or more, or 565 or more amino acids. In someembodiments, polypeptide fragments can comprise fewer than 30, fewerthan 45, fewer than 60, fewer than 75, fewer than 90, fewer than 120,fewer than 150, fewer than 180, fewer than 210, fewer than 240, fewerthan 270, fewer than 300, fewer than 360, fewer than 420, fewer than480, fewer than 540, or fewer than 565 amino acids. Preferably, thepolypeptide fragments are fragments of SEQ ID NOS:2, 4, 8, 10, 12, or14, and more preferably fragments of SEQ ID NOS:2, 6, 10, 12, or 14, andeven more preferably fragments of SEQ ID NOS:2, 10, or 14.

The determination of a fragment eliciting an immune response in a mammalsubstantially similar to that of the non-fragment for at least oneinfluenza subtype can be readily determined by one of ordinary skill.The fragment can be analyzed to contain at least one, preferably more,antigenic epitopes as provided by a publicly available database, such asthe Los Alamos National Laboratory's Influenza Sequence Database. Inaddition, immune response studies can be routinely assessed using miceand HI titers and ELISpots analysis, such as that shown in the Examplesbelow.

According to some embodiments of the invention, methods of inducing oreliciting an immune response in mammals against a plurality of influenzaviruses comprise administering to the mammals: a) the influenza strainH5N1 consensus HA protein, functional fragments thereof, or expressiblecoding sequences thereof; and b) one or more isolated encoding nucleicacid molecules provided herein, protein encoded by such nucleic acidmolecules, or fragments thereof.

According to some embodiments of the invention, methods of inducing oreliciting an immune response in mammals against a plurality of influenzaviruses comprise administering to the mammals: a) the influenza strainH1N1 and influenza strain H5N1 consensus NA protein, functionalfragments thereof, or expressible coding sequences thereof; and b) oneor more isolated encoding nucleic acid molecules provided herein,protein encoded by such nucleic acid molecules, or fragments thereof.

According to some embodiments of the invention, methods of inducing oreliciting an immune response in mammals against a plurality of influenzaviruses comprise administering to the mammals: a) the influenza strainH1N1 and influenza strain H5N1 consensus M1 protein, functionalfragments thereof, or expressible coding sequences thereof; and b) oneor more isolated encoding nucleic acid molecules provided herein,protein encoded by such nucleic acid molecules, or fragments thereof.

According to some embodiments of the invention, methods of inducing oreliciting an immune response in mammals against a plurality of influenzaviruses comprise administering to the mammals: a) the influenza strainH5N1 M2E-NP consensus protein, functional fragments thereof, orexpressible coding sequences thereof; and b) one or more isolatedencoding nucleic acid molecules provided herein, protein encoded by suchnucleic acid molecules, or fragments thereof.

According to some embodiments of the invention, methods of inducing oreliciting an immune response in mammals against a plurality of influenzaviruses comprise administering to the mammals: a) the influenza strainH1N1 HA consensus protein, functional fragments thereof, or expressiblecoding sequences thereof; and b) one or more isolated encoding nucleicacid molecules provided herein, protein encoded by such nucleic acidmolecules, or fragments thereof.

According to some embodiments of the invention, methods of inducing oreliciting an immune response in mammals against a plurality of influenzaviruses comprise administering to the mammals: a) the influenza strainH3N1 HA consensus protein, functional fragments thereof, or expressiblecoding sequences thereof; and b) one or more isolated encoding nucleicacid molecules provided herein, protein encoded by such nucleic acidmolecules, or fragments thereof.

In some embodiments of the invention, the vaccines of the inventioninclude at least two of the following sequences: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, and SEQ ID NO:14, or any combination of two or more sequencesfrom the aforementioned list.

Vaccines

In some embodiments, the invention provides improved vaccines byproviding proteins and genetic constructs that encode proteins withepitopes that make them particularly effective as immunogens againstwhich immune responses can be induced. Accordingly, vaccines can beprovided to induce a therapeutic or prophylactic immune response.

According to some embodiments of the invention, a vaccine according tothe invention is delivered to an individual to modulate the activity ofthe individual's immune system and thereby enhance the immune response.When a nucleic acid molecule that encodes the protein is taken up bycells of the individual the nucleotide sequence is expressed in thecells and the protein are thereby delivered to the individual. Aspectsof the invention provide methods of delivering the coding sequences ofthe protein on nucleic acid molecule such as plasmid.

According to some aspects of the present invention, compositions andmethods are provided which prophylactically and/or therapeuticallyimmunize an individual.

When taken up by a cell, the DNA plasmids can stay present in the cellas separate genetic material. Alternatively, RNA may be administered tothe cell. It is also contemplated to provide the genetic construct as alinear minichromosome including a centromere, telomeres and an origin ofreplication. Genetic constructs include regulatory elements necessaryfor gene expression of a nucleic acid molecule. The elements include: apromoter, an initiation codon, a stop codon, and a polyadenylationsignal. In addition, enhancers are often required for gene expression ofthe sequence that encodes the target protein or the immunomodulatingprotein. It is necessary that these elements be operable linked to thesequence that encodes the desired proteins and that the regulatoryelements are operably in the individual to whom they are administered.

Initiation codons and stop codon are generally considered to be part ofa nucleotide sequence that encodes the desired protein. However, it isnecessary that these elements are functional in the mammals to whom thenucleic acid construct is administered. The initiation and terminationcodons must be in frame with the coding sequence.

Promoters and polyadenylation signals used must be functional within thecells of the individual.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to promoters from simian virus 40 (SV40), mousemammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV)such as the bovine immunodeficiency virus (BIV) long terminal repeat(LTR) promoter, Moloney virus, avian leukosis virus (ALV),cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr virus (EBV), Rous sarcoma virus (RSV) as well as promoters fromhuman genes such as human actin, human myosin, human hemoglobin, humanmuscle creatine and human metalothionein; in other embodiments,promoters can be tissue specific promoters, such as muscle or skinspecific promoters, natural or synthetic. Examples of such promoters aredescribed in US patent application publication no. US20040175727, whichis incorporated hereby in its entirety.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to SV40 polyadenylation signals, LTRpolyadenylation signals, bovine growth hormone (bGH) polyadenylationsignals, human growth hormone (hGH) polyadenylation signals, and humanβ-globin polyadenylation signals. In particular, the SV40polyadenylation signal that is in pCEP4 plasmid (Invitrogen, San Diego,Calif.), referred to as the SV40 polyadenylation signal, can be used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to: human actin, human myosin, humanhemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

Genetic constructs can be provided with mammalian origin of replicationin order to maintain the construct extrachromosomally and producemultiple copies of the construct in the cell. Plasmids pVAX1, pCEP4 andpREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region whichproduces high copy episomal replication without integration.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the cells theconstruct is administered into. Moreover, codons that encode saidprotein may be selected which are most efficiently transcribed in thehost cell. One having ordinary skill in the art can produce DNAconstructs that are functional in the cells.

In some embodiments, nucleic acid constructs may be provided in whichthe coding sequences for the proteins described herein are linked to IgEsignal peptide. In some embodiments, proteins described herein arelinked to IgE signal peptide.

In some embodiments for which protein is used, for example, one havingordinary skill in the art can, using well known techniques, can produceand isolate proteins of the invention using well known techniques. Insome embodiments for which protein is used, for example, one havingordinary skill in the art can, using well known techniques, inserts DNAmolecules that encode a protein of the invention into a commerciallyavailable expression vector for use in well known expression systems.For example, the commercially available plasmid pSE420 (Invitrogen, SanDiego, Calif.) may be used for production of protein in Escherichia coli(E. coli). The commercially available plasmid pYES2 (Invitrogen, SanDiego, Calif.) may, for example, be used for production in Saccharomycescerevisiae strains of yeast. The commercially available MAXBAC™ completebaculovirus expression system (Invitrogen, San Diego, Calif.) may, forexample, be used for production in insect cells. The commerciallyavailable plasmid pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.) may,for example, be used for production in mammalian cells such as Chinesehamster ovary (CHO) cells. One having ordinary skill in the art can usethese commercial expression vectors and systems or others to produceprotein by routine techniques and readily available starting materials.(See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual,Second Ed. Cold Spring Harbor Press (1989)). Thus, the desired proteinscan be prepared in both prokaryotic and eukaryotic systems, resulting ina spectrum of processed forms of the protein.

One having ordinary skill in the art may use other commerciallyavailable expression vectors and systems or produce vectors using wellknown methods and readily available starting materials. Expressionsystems containing the requisite control sequences, such as promotersand polyadenylation signals, and preferably enhancers are readilyavailable and known in the art for a variety of hosts. See e.g.,Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. ColdSpring Harbor Press (1989). Genetic constructs include the proteincoding sequence operably linked to a promoter that is functional in thecell line, or cells of targeted tissue, into which the constructs aretransfected. Examples of constitutive promoters include promoters fromcytomegalovirus (CMV) or SV40. Examples of inducible promoters includemouse mammary leukemia virus or metallothionein promoters. Those havingordinary skill in the art can readily produce genetic constructs usefulfor transfecting cells with DNA that encodes protein of the inventionfrom readily available starting materials. The expression vectorincluding the DNA that encodes the protein is used to transform thecompatible host which is then cultured and maintained under conditionswherein expression of the foreign DNA takes place.

The protein produced is recovered from the culture, either by lysing thecells or from the culture medium as appropriate and known to those inthe art. One having ordinary skill in the art can, using well knowntechniques, isolate protein that is produced using such expressionsystems. The methods of purifying protein from natural sources usingantibodies which specifically bind to a specific protein as describedabove may be equally applied to purifying protein produced byrecombinant DNA methodology.

In addition to producing proteins by recombinant techniques, automatedpeptide synthesizers may also be employed to produce isolated,essentially pure protein. Such techniques are well known to those havingordinary skill in the art and are useful if derivatives which havesubstitutions not provided for in DNA-encoded protein production.

The nucleic acid molecules may be delivered using any of several wellknown technologies including DNA injection (also referred to as DNAvaccination) with and without in vivo electroporation, liposomemediated, nanoparticle facilitated, recombinant vectors such asrecombinant adenovirus, recombinant adenovirus associated virus andrecombinant vaccinia. Preferably, the nucleic acid molecules such as theDNA plasmids described herein are delivered via DNA injection and alongwith in vivo electroporation.

Routes of administration include, but are not limited to, intramuscular,intransally, intraperitoneal, intradermal, subcutaneous, intravenous,intraarterially, intraoccularly and oral as well as topically,transdermally, by inhalation or suppository or to mucosal tissue such asby lavage to vaginal, rectal, urethral, buccal and sublingual tissue.Preferred routes of administration include intramuscular,intraperitoneal, intradermal and subcutaneous injection. Geneticconstructs may be administered by means including, but not limited to,traditional syringes, needleless injection devices, “microprojectilebombardment gone guns”, or other physical methods such aselectroporation (“EP”), “hydrodynamic method”, or ultrasound.

Examples of electroporation devices and electroporation methodspreferred for facilitating delivery of the DNA vaccines of the presentinvention, include those described in U.S. Pat. No. 7,245,963 byDraghia-Akli, et al., U.S. Patent Pub. 2005/0052630 submitted by Smith,et al., the contents of which are hereby incorporated by reference intheir entirety. Also preferred, are electroporation devices andelectroporation methods for facilitating delivery of the DNA vaccinesprovided in co-pending and co-owned U.S. patent application Ser. No.11/874,072, filed Oct. 17, 2007, which claims the benefit under 35 USC119(e) to U.S. Provisional Application Ser. Nos. 60/852,149, filed Oct.17, 2006, and 60/978,982, filed Oct. 10, 2007, all of which are herebyincorporated in their entirety. Preferable, the electroporation deviceis the CELLECTRA™ device (VGX Pharmaceuticals, Blue Bell, Pa.),including the intramuscular (IM) and intradermal (ID) models.

U.S. Pat. No. 7,245,963 by Draghia-Akli, et al. describes modularelectrode systems and their use for facilitating the introduction of abiomolecule into cells of a selected tissue in a body or plant. Themodular electrode systems comprise a plurality of needle electrodes; ahypodermic needle; an electrical connector that provides a conductivelink from a programmable constant-current pulse controller to theplurality of needle electrodes; and a power source. An operator cangrasp the plurality of needle electrodes that are mounted on a supportstructure and firmly insert them into the selected tissue in a body orplant. The biomolecules are then delivered via the hypodermic needleinto the selected tissue. The programmable constant-current pulsecontroller is activated and constant-current electrical pulse is appliedto the plurality of needle electrodes. The applied constant-currentelectrical pulse facilitates the introduction of the biomolecule intothe cell between the plurality of electrodes. The entire content of U.S.Pat. No. 7,245,963 is hereby incorporated by reference.

U.S. Patent Pub. 2005/0052630 submitted by Smith, et al. describes anelectroporation device which may be used to effectively facilitate theintroduction of a biomolecule into cells of a selected tissue in a bodyor plant. The electroporation device comprises an electro-kinetic device(“EKD device”) whose operation is specified by software or firmware. TheEKD device produces a series of programmable constant-current pulsepatterns between electrodes in an array based on user control and inputof the pulse parameters, and allows the storage and acquisition ofcurrent waveform data. The electroporation device also comprises areplaceable electrode disk having an array of needle electrodes, acentral injection channel for an injection needle, and a removable guidedisk. The entire content of U.S. Patent Pub. 2005/0052630 is herebyincorporated by reference.

The electrode arrays and methods described in U.S. Pat. No. 7,245,963and U.S. Patent Pub. 2005/0052630 are adapted for deep penetration intonot only tissues such as muscle, but also other tissues or organs.Because of the configuration of the electrode array, the injectionneedle (to deliver the biomolecule of choice) is also insertedcompletely into the target organ, and the injection is administeredperpendicular to the target issue, in the area that is pre-delineated bythe electrodes The electrodes described in U.S. Pat. No. 7,245,963 andU.S. Patent Pub. 2005/005263 are preferably 20 mm long and 21 gauge.

The following is an example of methods of the present invention, and isdiscussed in more detail in the patent references discussed above:electroporation devices can be configured to deliver to a desired tissueof a mammal a pulse of energy producing a constant current similar to apreset current input by a user. The electroporation device comprises anelectroporation component and an electrode assembly or handle assembly.The electroporation component can include and incorporate one or more ofthe various elements of the electroporation devices, including:controller, current waveform generator, impedance tester, waveformlogger, input element, status reporting element, communication port,memory component, power source, and power switch. The electroporationcomponent can function as one element of the electroporation devices,and the other elements are separate elements (or components) incommunication with the electroporation component. In some embodiments,the electroporation component can function as more than one element ofthe electroporation devices, which can be in communication with stillother elements of the electroporation devices separate from theelectroporation component. The present invention is not limited by theelements of the electroporation devices existing as parts of oneelectromechanical or mechanical device, as the elements can function asone device or as separate elements in communication with one another.The electroporation component is capable of delivering the pulse ofenergy that produces the constant current in the desired tissue, andincludes a feedback mechanism. The electrode assembly includes anelectrode array having a plurality of electrodes in a spatialarrangement, wherein the electrode assembly receives the pulse of energyfrom the electroporation component and delivers same to the desiredtissue through the electrodes. At least one of the plurality ofelectrodes is neutral during delivery of the pulse of energy andmeasures impedance in the desired tissue and communicates the impedanceto the electroporation component. The feedback mechanism can receive themeasured impedance and can adjust the pulse of energy delivered by theelectroporation component to maintain the constant current.

In some embodiments, the plurality of electrodes can deliver the pulseof energy in a decentralized pattern. In some embodiments, the pluralityof electrodes can deliver the pulse of energy in the decentralizedpattern through the control of the electrodes under a programmedsequence, and the programmed sequence is input by a user to theelectroporation component. In some embodiments, the programmed sequencecomprises a plurality of pulses delivered in sequence, wherein eachpulse of the plurality of pulses is delivered by at least two activeelectrodes with one neutral electrode that measures impedance, andwherein a subsequent pulse of the plurality of pulses is delivered by adifferent one of at least two active electrodes with one neutralelectrode that measures impedance.

In some embodiments, the feedback mechanism is performed by eitherhardware or software. Preferably, the feedback mechanism is performed byan analog closed-loop circuit. Preferably, this feedback occurs every 50μs, 20 μs, 10 μs or 1 μs, but is preferably a real-time feedback orinstantaneous (i.e., substantially instantaneous as determined byavailable techniques for determining response time). In someembodiments, the neutral electrode measures the impedance in the desiredtissue and communicates the impedance to the feedback mechanism, and thefeedback mechanism responds to the impedance and adjusts the pulse ofenergy to maintain the constant current at a value similar to the presetcurrent. In some embodiments, the feedback mechanism maintains theconstant current continuously and instantaneously during the delivery ofthe pulse of energy.

A pharmaceutically acceptable excipient can include such functionalmolecules as vehicles, adjuvants, carriers or diluents, which are knownand readily available to the public. Preferably, the pharmaceuticallyacceptable excipient is an adjuvant or transfection facilitating agent.In some embodiments, the nucleic acid molecule, or DNA plasmid, isdelivered to the cells in conjunction with administration of apolynucleotide function enhancer or a genetic vaccine facilitator agent(or transfection facilitating agent). Polynucleotide function enhancersare described in U.S. Pat. Nos. 5,593,972, 5,962,428 and InternationalApplication Serial Number PCT/US94/00899 filed Jan. 26, 1994, which areeach incorporated herein by reference. Genetic vaccine facilitatoragents are described in U.S. Ser. No. 021,579 filed Apr. 1, 1994, whichis incorporated herein by reference. The transfection facilitating agentcan be administered in conjunction with nucleic acid molecules as amixture with the nucleic acid molecule or administered separatelysimultaneously, before or after administration of nucleic acidmolecules. Examples of transfection facilitating agents includes surfaceactive agents such as immune-stimulating complexes (ISCOMS), Freundsincomplete adjuvant, LPS analog including monophosphoryl lipid A,muramyl peptides, quinone analogs and vesicles such as squalene andsqualene, and hyaluronic acid may also be used administered inconjunction with the genetic construct. In some embodiments, the DNAplasmid vaccines may also include a transfection facilitating agent suchas lipids, liposomes, including lecithin liposomes or other liposomesknown in the art, as a DNA-liposome mixture (see for example W09324640),calcium ions, viral proteins, polyanions, polycations, or nanoparticles,or other known transfection facilitating agents. Preferably, thetransfection facilitating agent is a polyanion, polycation, includingpoly-L-glutamate (LGS), or lipid.

In some preferred embodiments, the DNA plasmids are delivered with anadjuvant that are genes for proteins which further enhance the immuneresponse against such target proteins. Examples of such genes are thosewhich encode other cytokines and lymphokines such as alpha-interferon,gamma-interferon, platelet derived growth factor (PDGF), TNFα, TNFβ,GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6,IL-10, IL-12, IL-18, MHC, CD80, CD86 and IL-15 including IL-15 havingthe signal sequence deleted and optionally including the signal peptidefrom IgE. Other genes which may be useful include those encoding: MCP-1,MIP-1α, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34,GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2,ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40,CD40L, vascular growth factor, fibroblast growth factor, IL-7, nervegrowth factor, vascular endothelial growth factor, Fas, TNF receptor,Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5,KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1,Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1,JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND,NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 andfunctional fragments thereof.

The pharmaceutical compositions according to the present inventioncomprise DNA quantities of from about 1 nanogram to 100 milligrams;about 1 microgram to about 10 milligrams; or preferably about 0.1microgram to about 10 milligrams; or more preferably about 1 milligramto about 2 milligram. In some preferred embodiments, pharmaceuticalcompositions according to the present invention comprise about 5nanogram to about 1000 micrograms of DNA. In some preferred embodiments,the pharmaceutical compositions contain about 10 nanograms to about 800micrograms of DNA. In some preferred embodiments, the pharmaceuticalcompositions contain about 0.1 to about 500 micrograms of DNA. In somepreferred embodiments, the pharmaceutical compositions contain about 1to about 350 micrograms of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 25 to about 250 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 100 to about 200 microgram DNA.

The pharmaceutical compositions according to the present invention areformulated according to the mode of administration to be used. In caseswhere pharmaceutical compositions are injectable pharmaceuticalcompositions, they are sterile, pyrogen free and particulate free. Anisotonic formulation is preferably used. Generally, additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitoland lactose. In some cases, isotonic solutions such as phosphatebuffered saline are preferred. Stabilizers include gelatin and albumin.In some embodiments, a vasoconstriction agent is added to theformulation. In some embodiments, a stabilizing agent that allows theformulation to be stable at room or ambient temperature for extendedperiods of time, such as LGS or other polycations or polyanions is addedto the formulation.

In some embodiments, methods of eliciting an immune response in mammalsagainst a consensus influenza antigen include methods of inducingmucosal immune responses. Such methods include administering to themammal one or more of CTACK protein, TECK protein, MEC protein andfunctional fragments thereof or expressible coding sequences thereof incombination with an DNA plasmid including a consensus influenza antigen,described above. The one or more of CTACK protein, TECK protein, MECprotein and functional fragments thereof may be administered prior to,simultaneously with or after administration of the DNA plasmid influenzavaccines provided herein. In some embodiments, an isolated nucleic acidmolecule that encodes one or more proteins of selected from the groupconsisting of: CTACK, TECK, MEC and functional fragments thereof isadministered to the mammal.

EXAMPLES

The present invention is further illustrated in the following Examples.It should be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Preferably the DNA formulations for use with a muscle or skin EP devicedescribed herein have high DNA concentrations, preferably concentrationsthat include milligram to tens of milligram quantities, and preferablytens of milligram quantities, of DNA in small volumes that are optimalfor delivery to the skin, preferably small injection volume, ideally25-200 microliters (μL). In some embodiments, the DNA formulations havehigh DNA concentrations, such as 1 mg/mL or greater (mg DNA/volume offormulation). More preferably, the DNA formulation has a DNAconcentration that provides for gram quantities of DNA in 200 μL offormula, and more preferably gram quantities of DNA in 100 μL offormula.

The DNA plasmids for use with the EP devices of the present inventioncan be formulated or manufactured using a combination of known devicesand techniques, but preferably they are manufactured using an optimizedplasmid manufacturing technique that is described in a commonly owned,co-pending U.S. provisional application U.S. Ser. No. 60/939,792, whichwas filed on May 23, 2007. In some examples, the DNA plasmids used inthese studies can be formulated at concentrations greater than or equalto 10 mg/mL. The manufacturing techniques also include or incorporatevarious devices and protocols that are commonly known to those ofordinary skill in the art, in addition to those described in U.S. Ser.No. 60/939,792, including those described in a commonly owned patent,U.S. Pat. No. 7,238,522, which issued on Jul. 3, 2007. The highconcentrations of plasmids used with the skin EP devices and deliverytechniques described herein allow for administration of plasmids intothe ID/SC space in a reasonably low volume and aids in enhancingexpression and immunization effects. The commonly owned application andpatent, U.S. Ser. No. 60/939,792 and U.S. Pat. No. 7,238,522,respectively, are hereby incorporated in their entirety.

Example 1 Plasmid Constructs

A ubiquitous cytomegalovirus (CMV) promoter drives the expression ofhuman secreted embryonic alkaline phosphatase (SEAP) reporter transgeneproduct in the pCMV-SEAP vector. Plasmids were obtained using acommercially available kit (Qiagen Inc., Chatsworth, Calif.). Endotoxinlevels were at less than 0.01 EU/μg, as measured by Kinetic ChromagenicLAL (Endosafe, Charleston, S.C.). Consensus HA and NA constructs weregenerated by analyzing primary virus sequences from 16H5 viruses thathave proven fatal to humans in recent years, and over 40 human N1viruses. These sequences were downloaded from the Los Alamos NationalLaboratory's Influenza Sequence Database. After generating the consensussequences, the constructs were optimized for mammalian expression,including the addition of a Kozak sequence, codon optimization, and RNAoptimization. These constructs were then subcloned into the pVAX vector(Invitrogen, Carlsbad, Calif.). Unless indicated otherwise, plasmidpreparations were diluted in sterile water and formulated 1%weight/weight with poly-L-glutamate sodium salt (LGS) (MW=10.5 kDaaverage)(Sigma, St. Louis, Mo.), further HPLC purified at VGXPharmaceuticals, Immune Therapeutics Division (The Woodlands, Tex.).

Example 2 Treatment of Pigs

Pigs were divided into 10 groups×4 pigs per group for a total of 40 pigs(Table 1). Pigs were acclimated for 4 days, weighed and ear-tagged. OnStudy Day 0, pigs were weighed, bled and anesthetized using acombination pre-anesthetic for pigs—ketamine—(20 mg/kg), xylazine—(2.2mg/kg) and atropine (0.04 mg/kg), and then anesthetized using isoflurane(induction at 5%, maintenance at 2-3%). Pigs (n=4/group) were injectedwith 0.6 mL of CMV-HA (a pVAX based construct that expresses a consensusH5 antigen), CMV-NA (a pVAX based construct that expresses a consensusN1 antigen), and CMV-SEAP (a construct expressing the reporter genesecreated ambryonic alkaline phosphatase, SEAP) plasmid (the last oneadded to increase plasmid concentration, and viscosity of the solutionfor the “muscle damage” assessment)+1.0% wt/wt LGS at varying plasmidconcentrations and current intensities. The plasmids were preparedaccording to the materials and methods provided in Example 1. After 4s,animals were electroporated using the adaptive constant currentCELLECTRA™ intramuscular (IM) system (VGX Pharmaceuticals, Blue Bell,Pa.) equipped with 5 needle electrodes and operated with the followingpulse parameters: 52 millisecond pulses, 1 second between pulses, 3pulses with varying current (0.1, 0.3 and 0.5 A).

TABLE 1 Groups for the pig vaccine experiment Con- Conc struct TotalInjec- (mg/ (mg)/ Dose tion Group Plasmid mL) pig (mg/pig) Volume A n 1HA, NA, 10 2 6 600 μl 0.5 4 SEAP 2 HA, NA, 4 0.8 2.4 600 μl 0.5 4 SEAP 3HA, NA, 1.5 0.1 0.3 600 μl 0.5 4 SEAP 4 HA, NA, 10 2 6 600 μl 0.3 4 SEAP5 HA, NA, 4 0.8 2.4 600 μl 0.3 4 SEAP 6 HA, NA, 1.5 0.1 0.3 600 μl 0.3 4SEAP 7 HA, NA, 10 2 6 600 μl 0.1 4 SEAP 8 HA, NA, 4 0.8 2.4 600 μl 0.1 4SEAP 9 HA, NA, 1.5 0.1 0.3 600 μl 0.1 4 SEAP 10 None N/A N/A N/A N/A N/A4The area surrounding each injection site was tattooed for rapididentification for biopsy at Days 14 and 35 post-injection.

Pigs were allowed to recover from anesthesia and were closely monitoredfor 24 hours to ensure full recovery. Any pigs that did not fullyrecover within 2 to 3 hours post-treatment were noted. Pigs were weighedand bled on Day 10, Day 21 and Day 35. The pigs were administered asecond vaccination on Day 21. Blood was collected in 2 purple top tubes,1.0 mL for CBC and differentials (Antech Diagnostics, Irvine, Calif.);10 mL for IFN-γ ELISpots against HA and NA antigens, and separate falcontubes which were allowed to clot and centrifuged to isolate serum thenaliquoted into tubes on ice. On Day 35, all pigs were exsanguinatedunder surgical plane of anesthesia and needle punch biopsies of theinjection sites were taken for histology.

Hemagglutination Inhibition (HI) Assay

Pig sera were treated with receptor destroying enzyme (RDE) by dilutingone part serum with three parts enzyme and incubated overnight in 37° C.water bath. The enzyme was inactivated by 30 min incubation at 56° C.followed by addition of six parts PBS for a final dilution of 1/10. HIassays were performed in V-bottom 96-well microtiter plates, using fourHA units of virus and 1% horse red blood cells as previously described(Stephenson, I., et al., Virus Res., 103(1-2):91-5 (July 2004)).

The highest titers as demonstrated by the HI assay (FIG. 2) were foundin sera from the group administered 2 mg of HA-expressing plasmid at acurrent setting of 0.5 A (120±40; P=0.11 versus 2 mg/0.3 A and P=0.02versus 2 mg/0.1 A); the titers decreased with the intensity of theelectric field for the group that received 2 mg of each plasmid; ifeither plasmid quantity of current were decreased thereafter, the titerswere more variable, and non-different between groups.

The HI titers were highest in the group administered 2 mg ofHA-expressing plasmid and electroporated at 0.5 A. Furthermore, thetiters decline with descending plasmid doses in the group electroporatedat 0.5 A, and with the intensity of the electric field. The lowerplasmid quantities or lower current intensities appeared to increase theintra-group variability.

HA and NA IFN-γ ELISpots

ELISpot were performed as previously described using IFN-γ capture anddetection antibodies (MabTech, Sweden) (Boyer J D, et al., J MedPrimatol, 34(5-6):262-70 (October 2005)). Antigen-specific responseswere determined by subtracting the number of spots in the negativecontrol wells from the wells containing peptides. Results are shown asthe mean value (spots/million splenocytes) obtained for triplicatewells.

The group administered 2 mg of each plasmid (for a total of 4 mg) at acurrent setting of 0.3 A attained the highest cellular immune responseas measured by the IFN-γ ELISpot of 537±322 SFU per million cells. Theaverage responses of all other groups were within background levels ofthe assay. The individual ELISpot responses of two animals attaining thehighest cellular immune response are highlighted in FIG. 3.

CBC Results

Lymphocytes reached the highest levels at Day 21 of the study and in thegroups administered the highest dose of vaccines, regardless of currentsetting, although the groups with the highest dose (4 mg of totalplasmid, 2 mg each) and highest current setting (0.5 A) demonstrateshighest lymphocyte response, 40% higher than controls (12670±1412 vs.7607±1603 lymphocyte counts/100 blood, respectively; P<0.002).

Muscle Histopathology

The injection sites were identified and punch biopsies were taken atDays 14 and 35 post-treatment after the pigs were exsanguinated. Thetissues were fixed in buffered formalin for 24 hours then washed 3× inPBS and stored in 70% alcohol. The biopsy samples were submitted toAntech Diagnostics where they were processed and sections stained withhematoxylin and eosin (H&E). All the slides were evaluated by a singleboard-certified pathologist who scored them 0 to 5 for pathologicalcriteria (Table 2) in various tissue layers (Table 3). The mean scorewas calculated for each group at each time point.

TABLE 2 Biopsy pathology scoring parameters Score Criteria 0 Notpresent, no inflammatory cells 1 Minimal, 1-20 inflammatory cells/100 ×high-powered field (HPF) 2 Mild, 21-40 inflammatory cells/100 × HPF 3Moderate, 41-75 inflammatory cells/100 × HPF 4 Moderate toMarked/Severe, 76-100 inflammatory cells/100 × HPF 5 Marked Severe, >100inflammatory cells/100 × HPF

TABLE 3 Biopsy tissue layers and pathological parameters AnatomyLocation Pathology Parameter Dermal Superficial neovascularizationDermal Pylogranulomatous inflammation Dermal Overlying erosion &inflammatory crusting Dermal Focal fibrosis SubcutaneousPylogranulomatous inflammation with intralesional collagen necrosisSubcutaneous Lymphacytic and plasmalytic inflammation Skeletal muscleLymphacytic and plasmalytic and eosinophilic inflammation Skeletalmuscle Myocyte degeneration/necrosis Skeletal muscle Fibrosis

The histopathology was scored from the muscle biopsy (FIG. 4A) at 14 and35 days after plasmid injection and EP based on a 0 to 5 scale criteria(Table 2). Overall pathology scores following electroporation declinedin the tissue layers (Table 3) from Day 14 to Day 35. The group thatreceived 6 mg of total plasmid at 0.3 A settings exhibited the highesttotal pathology scores at Day 14 (18.3±6.4, P<0.0002 versus control),correlating with the highest average lymphocyte responses. All pathologyscores at Day 35 approached levels of non-treated control levels (rangeof 6.67 to 4.25). Nevertheless, when the muscle necrosis and fibrosis(typically associated with the EP procedure) (Gronevik E, et al., J GeneMed, 7(2):218-27 (2005 February)). were analyzed separately (FIG. 4B),the scores ranged between 1 and 2, with no difference between groups orbetween treated groups and controls, while the higher scores wereassociated with lymphatic, plasmacytic or eosinophilic inflammation dueto immune responses. Significantly, these scores also declined from day14 to day 35 post-treatment.

Data Analysis

Data were analyzed using Microsoft Excel Statistics package. Valuesshown in the figures are the mean±SEM. Specific values were obtained bycomparison using one-way ANOVA and subsequent t-test. A value of p<0.05was set as the level of statistical significance.

Example 3 Treatment of Ferrets

Twenty male ferrets (Triple F Farms, Sayre, Pa.), 4-6 months of age orat least 1 kg body weight, were used in this study and housed atBIOQUAL, Inc. (Rockville, Md.). The ferret study design is in Table 4.Animals were allowed to acclimate for two weeks prior to the study.Animals were immunized (under anesthesia) at Week 0, 4, and 9. Blood wasdrawn every 2 weeks. After the third immunization, animals were movedinto a BSL-3 facility and challenged at Week 13 with a very potentstrain of avian influenza (H5N1) and then followed for two more weekspost-challenge. For two weeks after challenge, animals were monitoreddaily, and body weights, temperature and clinical scores were recorded.Activity level was monitored and recorded; death were documented.

This study tested the efficacy of HA, NA and M2e-NP DNA vaccinedelivered IM followed by electroporation using the CELLECTRA™ adaptiveconstant current electroporation intramuscular (IM) system (VGXPharmaceuticals, Blue Bell, Pa.) in an influenza challenge model inferrets. The DNA plasmids were prepared according to the materials andmethods provided in Example 1. As outlined in Table 4, animals in Groups2, 3 and 4 received 0.2 mg of the respective influenza plasmid vaccine.In order to correct for dose, groups which received 1 plasmid vaccine(Groups 2 and 3) or no vaccine (control Group 1), the difference wasmade up by pVAX empty vector such that all animals in every groupreceived a total dose of 0.6 mg of plasmid. The conditions ofelectroporation were, using a 5 needle electrode array: 0.5 Amps, 52msec pulse width, 1 sec between pulses, 4 sec delay between injectionand electroporation.

TABLE 4 Groups for the Influenza challenge experiment in ferrets TotalVaccine vaccine in Dose (mg) Total Group Plasmids/Antigens per Plasmidvolume n 1 None (pVAX only)   0 mg 0.6 mg 4 in 0.6 mL 2 H5 + pVAX 0.2 mg0.6 mg 4 in 0.6 mL 3 NA + pVAX 0.2 mg 0.6 mg 4 in 0.6 mL 4 H5, NA,M2e-NP 0.2 mg 0.6 mg 4 in 0.6 mLHemagglutination Inhibition (HI) Assay

Sera were treated with receptor destroying enzyme (RDE) by diluting onepart serum with three parts enzyme and incubated overnight in 37° C.water bath. The enzyme was inactivated by 30 min incubation at 56° C.followed by addition of six parts PBS for a final dilution of 1/10. HIassays were performed in V-bottom 96-well microtiter plates, using fourHA units of virus and 1% horse red blood cells as previously described(Stephenson, I., et al., Virus Res., 103(1-2):91-5 (July 2004)). Theviruses used for the HI assay are reassortant strains we obtained fromthe Center for Disease Control: A/Viet/1203/2004(H5N1)/PR8-IBCDC-RG(clade 1 virus) and A/Indo/05/2005 (H5N1)/PR8-IBCDC-RG2 (clade 2 virus).The ferret model of influenza infection is considered to be morereflective of human disease and a more rigorous challenge model. Ferretsexhibit similar symptoms to humans infected with influenza and similartissue tropism with regards to human and avian influenza viruses. Serumcollected at different time points throughout the study was used todetect HI activity against H5N1 viruses. As shown in FIG. 7, both groupscontaining the consensus H5 specific HA construct attained protectivelevels of antibody (>1:40) after two immunizations and were also able toinhibit a clade 2 H5N1 virus. In other words, the HI assay was positiveagainst both viral strains despite the consensus HA strain was based onclade 1 viruses.

Data Analysis

Data were analyzed using Microsoft Excel Statistics package. Valuesshown in the figures are the mean±SEM. Specific values were obtained bycomparison using one-way ANOVA and subsequent t-test. A value of p<0.05was set as the level of statistical significance.

Ferret Influenza Challenge

The results of the influenza challenge are depicted in FIGS. 5 and 6.Control animals lost 25% of their body weight on average post-challenge(FIG. 5), while animals vaccinated with HA (Group 1) or HA+M2e-NP+NA(Group 4) lost between 9 and 10% (*P<0.004 versus controls). Bodytemperatures were elevated in control animals until all control animalswere either found dead or euthanized by Day 8 (FIG. 6). All animalsvaccinated, regardless of which vaccine regimen, survived the challengeand showed fewer signs of infection as compared to the control animalsas evidenced by their clinical scores (Table 5). Control animals worsenas far as clinical scores (nasal discharge, cough, lethargy), and diedbetween day 5 and day 7 post-challenge. As shown in Table 5, theseverity of the clinical scores in vaccinated animals was inverselycorrelated with the antibody titers (higher antibody titers, lowerclinical scores, better clinical outcome).

TABLE 5 Results for Challenged Ferrets Post-challenge Observations HITiters 3 wks Vaccines Ferret Day 1 Day 2 Day 3 Day 5 Day 6 Day 7 Day 8Day 9 Pre-challenge 891 0_1 1_1 1_1 0_1 0_1  1_1* <20 Control 890 0_10_1 1_1  0_1* <20 (pVAX) 877 0_1 0_1 1_1 0_2 2_3 FD <20 876 0_1 0_1 0_10_1 2_3  1_3* <20 878 0_1 0_1 1_1 0_1 0_1 0_1 0_1 0_1 40 H5 879 0_1 0_10_1 0_1 0_1 0_1 0_1 0_1 320 888 0_1 0_1 0_1 0_1 0_1 0_1 0_1 0_1 160 8890_1 0_1 0_1 0_1 0_1 0_1 0_1 0_1 320 881 0_1 0_1 1_0 0_1 0_1 1_1 0_1 0_1<20 M2-NP 880 0_1 0_1 0_0 0_1 0_1 0_1 0_1 0_1 <20 883 0_1 1_1 1_1 0_10_1 1_1 0_1 0_1 <20 882 0_1 0_1 1_1 0_1 0_1 1_2 0_2 0_1 <20 885 0_1 0_10_1 0_1 0_1 0_1 0_1 0_1 1280 H5 + M2 − 884 0_1 0_1 0_1 0_1 0_1 0_1 0_10_1 320 NP + NA 886 1_1 1_1 0_1 0_1 0_1 1_1 0_1 1_1 160 887 0_1 1_1 0_00_1 0_1 1_1 0_1 0_1 640 Table 5 Note: Clinical scores are depicted forthe post-challenge observation period. A “*” indicates the animal waseuthanized; FD = found dead. The first clinical score in each column isfor nasal symptoms: 0 = none; 1 = nasal discharge; 2 = breathing frommouth. The second score is for activity: 0 = sleeping; 1 = bright andalert; 2 = alert but nonresponsive; 3 = lethargic. The HI titers foreach animal measured 3 weeks pre-challenge are depicted for comparisonpurposes.

Example 4 Intradermal Delivery Comparisons with Intramuscular Deliveryin Primates

Rhesus macaques were immunized in these studies. Animals were acclimatedfor 2 months prior to the start of experiments. The study progressed asfollows: Week 0—performed 1st immunization (plasmid dose administration)and baseline bleed; Week 2 performed bleed; Week 3 performed 2ndimmunization (plasmid dose administration); Week 5 performed bleed; Week6 performed 3rd immunization (plasmid dose administration) and bleed;Week 8 performed bleed.

TABLE 6 Total Study DNA DNA Group Constructs Nr. Route of Admin Dose(mg) A DNA 6 + 9 5 IM CELLECTRA ™ EP 1 mg/ 2 Const B DNA 6 + 9 5 IDCELLECTRA ™ EP 1 mg/ 2 Const C DNA 1 + 6 + 5 IM Syringe 1 mg/ 4 9 + 10Const D Negative 5 N/A 0 Control DNA Construct # Encoding Antigen 1Non-influenza antigen control plasmid 6 Influenza H5 consensus 9Non-influenza antigen control plasmid 10 Non-influenza antigen controlplasmid

All plasmids were formulated at 10 mg/mL in water for injection+1% LGS,as described in previous examples, above, and mixed into a singlesolution PER STUDY GROUP(S) (Groups C, D, G, and H, in above table,Table 6). The correct injection volume for each group designated IMCELLECTRA™ EP (VGX Pharmaceuticals), ID CELLECTRA™ EP (VGXPharmaceuticals), and IM Syringe was calculated. For the IDadministration, if the required injection volume surpassed 100 μL persite, the formulation was split into a number of injection sites (2, 3,or 6 depending on how many total mg of vaccine were administered). Theanimals that received IM injection(s) were given the entire formulationin one single site.

The CELLECTRA™ adaptive constant current device used in the pigsexperiments, ferret experiments and nonhuman experiments described inthe Examples. The electroporation conditions were as following: for theIM injection and electroporation groups the conditions were: 0.5 Amps,52 msec/pulse, three pulses, 4 sec delay between plasmid injection andelectroporation. For the ID injection and electroporation groups theconditions were: 0.2 Amps, 52 msec/pulse, three pulses, 4 sec delaybetween plasmid injection and electroporation.

Hemagglutination Inhibition (HI) Assay—

monkey sera were treated with receptor destroying enzyme (RDE) bydiluting one part serum with three parts enzyme and incubated overnightin 37° C. water bath. The enzyme was inactivated by 30 min incubation at56° C. followed by addition of six parts PBS for a final dilution of1/10. HI assays were performed in V-bottom 96-well microtiter plates,using four HA units of virus and 1% horse red blood cells. The datapresented herein are the results after the second immunization (bleedcollected before the third immunization).

HI titers were measured three weeks after the second immunization. Theresults can be seen displayed in the graph in FIG. 8. Monkeys receivingthe HA plasmid vaccine via ID injection followed by electroporationdemonstrated more than twice the average titers of the IM+EP group andalmost three times the average titers of the IM group alone (*P<0.03).Non-treated controls did not exhibit any HI titers.

Example 5 Cross Protection in Primates

Using Delivery Method—ID Injection Followed by Electroporation (EP)

Studies in non-human primates with the influenza vaccine (including H5,NA and M2e-NP consensus antigens, see above) indicated that ID injectionfollowed by electroporation elicited higher antibody responses to thevaccine antigens than in IM injections. In one of our non-human primatestudies (NHP) animals were vaccinated per Table 7.

TABLE 7 Study design and conditions. Rhesus macaques were immunized atweeks 0, 4, and 8. Concentration Group n/group Antigen Delivery(mg/plasmid) EP Conditions 1 5 pVax (sham) IM 1 mg/construct 0.5 Amps, 3pulses, 52 msec, 1 sec between pulses 2 5 H5, NA, IM 1 mg/construct 0.5Amps, 3 pulses, 52 msec, 1 sec M2e-NP between pulses 3 5 M2e-NP IM 1mg/construct 0.5 Amps, 3 pulses, 52 msec, 1 sec between pulses 4 5 H5,NA, ID 1 mg/construct 0.2 Amps, 2 × 2 pulses, 52 msec, 1 sec M2e-NPbetween pulsesEach animal received three vaccinations, and HAI titers andmicroneutralization were performed for both the same clade andcross-clades. As shown, the consensus vaccine offered broad protectionnot only within the same clade, but also cross-clades. Results areincluded in Table 8.

TABLE 8 Results of hemagglutination (HAI) and microneutralizationassays. Clade 1 Clade 2.1 Clade 2.2 Clade 2.3.4 A/Vietnam A/IndonesiaA/Turkey A/Anhui HAI Assay 2nd immunization VGX-3400IM 160 (80-320)  36(20-80) 110 (0-320)^(4/5) 80 (40-160) VGX-3400ID 664 (40-1280) 120(20-320) 205 (0-320)^(4/5) 592 (40-1280) 3rd immunization VGX-3400IM 288(160-640) 32 (0-80)^(3/5) 36 (20-80) 84 (20-160) VGX-3400ID 416(160-640) 64 (0-160)^(2/5) 145 (20-320) 276 (20-640) Microneutralization3rd immunization VGX-3400IM 144 (40-360)  8 (0-40)^(1/5) 32 (0-80)^(2/5)88 (0-160)^(4/5) VGX-3400ID 740 (20-2560) 96 (0-320)^(3/5) 296(0-1280)^(3/5) 1172 (20-2560) Values presented indicate the mean titer,the range (in parenthesis) and the number of responders if less than 5/5(in superscript). Note: HAI titers > 1:20 are generally consideredseroprotective in the NHP model.

The needles in the ID electroporation device are much shorter (˜5 mm),of a lower gauge, and do not elicit muscle contractions or visible painresponses in the animals tested to date. Furthermore, the requiredelectric field for efficacious ID EP is lower than that required for anoptimum IM delivery. ID injection has been shown to elicit better immuneresponses to influenza vaccine antigens. (Holland D, et. al. (2008). JInf Dis. 198:650-58.) Usually, a lower dose is needed in vaccinesdelivered ID compared to IM delivery to achieve similar humoralresponses.

What is claimed is:
 1. A composition capable of expressing a consensusinfluenza antigen in a cell of a mammal, comprising: a first DNA plasmidcomprising: a nucleic acid sequence that encodes a consensushemagglutinin (HA), wherein said nucleic acid sequence that encodes theconsensus HA comprises a nucleotide sequence having 100% identity to SEQID NO:13, wherein the consensus HA elicits a protective immune responseacross different clades and within subtypes of the same clade upon whichthe consensus HA is based, and a promoter operably linked to the nucleicacid sequence that encodes the consensus HA that regulates expression ofthe consensus HA; and a second DNA plasmid comprising: a nucleic acidsequence that encodes a consensus antigen selected from the groupconsisting of a consensus neuraminidase (NA) and a consensus M2ectodomain-nucleoprotein (M2e-NP), wherein the consensus NA comprisesthe amino acid sequence of SEQ ID NO: 4 and wherein the consensus M2e-NPcomprises the amino acid sequence of SEQ ID NO: 8; and a promoteroperably linked to the nucleic acid sequence that encodes the consensusantigen that regulates expression of the consensus antigen.
 2. Acomposition comprising a nucleic acid sequence comprising SEQ ID NO:13,further comprising at least one selected from the group consisting of: anucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 4;and a nucleic acid sequence encoding the amino acid sequence of SEQ IDNO:
 8. 3. The composition of claim 2, wherein the composition comprisesa plasmid comprising the nucleic acid sequence comprising SEQ ID NO:13.4. The composition of claim 2, wherein the composition is apharmaceutical composition that further comprises a pharmaceuticalacceptable excipient.
 5. The composition of claim 2, wherein thecomposition further comprises a transfection facilitating agent.
 6. Thecomposition of claim 4, wherein the pharmaceutical composition furthercomprises a transfection facilitating agent.
 7. The composition of claim1, wherein the second DNA plasmid comprises: a nucleic acid sequencethat encodes the consensus NA, wherein the consensus NA comprises theamino acid sequence of SEQ ID NO: 4; and a promoter operably linked tothe nucleic acid sequence that encodes the consensus NA that regulatesexpression of the consensus NA; and wherein the composition furthercomprises a third DNA plasmid comprising: a nucleic acid sequence thatencodes the consensus M2e-NP, wherein the consensus M2e-NP comprises theamino acid sequence of SEQ ID NO: 8; and a promoter operably linked tothe nucleic acid sequence that encodes the consensus M2e-NP thatregulates expression of the consensus M2e-NP.
 8. The composition ofclaim 2, wherein the composition comprises a nucleic acid sequenceencoding the amino acid sequence of SEQ ID NO: 4; and a nucleic acidsequence encoding the amino acid sequence of SEQ ID NO: 8.